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Relationship between stability and bioluminescence color of firefly luciferase
Abstract and Figures
Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, Mg(2+) and molecular oxygen. The bioluminescence color of firefly luciferases is identified by the luciferase structure and assay conditions. Amongst different types of beetles, luciferase from Phrixotrix railroad worm (PhRE) with a unique additional residue (Arg353) naturally emits red bioluminescence color. By insertion of Arg356 in luciferase of Lampyris turkestanicus, corresponding to Arg353 in Phrixotrix hirtus, the color of the emitted light was changed to red. To understand the effect of this position on the bioluminescence color shift, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase. Comparison of mutants with native luciferase shows that mutation brought an increase in the content of secondary structure and globular compactness of (P. pylalis) luciferase. Comparative study of chemical denaturation of native and mutant luciferases by activity measurement, intrinsic and extrinsic fluorescence, circular dichroism, and DSC techniques revealed that insertion of positively charged residues (Arg, Lys) in the flexible loop (352-358) plays a significant role on the stability of (P. pyralis) luciferase and changes the light color to red.
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... Several mutations in green luciferases were reported to shift the colour of the emitted light, but only a few natural substitutions (including positions 339 and 353 in green-emitting luciferases) and one natural insertion (R353 in RE Ph ) have been identified by sequence comparisons. The individual contributions of multiple residues to the colour emission were tested by introducing synthetic mutations at these positions 31,51,52,54,56,58,115,116 . Nevertheless, mutations and their effect on the colour emitted by beetle luciferase reaction products are scattered throughout the available literature and also throughout the globular structure of luciferases (Fig. 6a). ...
... However, such strategies have not been the most common approach in the literature. Studies that have emphasized the comparison of luciferases that naturally emit particularly long-wavelength or short-wavelength light to discern key residues and effects responsible for colour shifting 31,50,54,56,115 are usually overlooked or not completely understood when the context of the work is not biochemical research. The disconnect goes both ways: most biochemical research (mainly mutational analysis) does not usually take into account the vast amount of fine spectroscopic data available. ...
... Although shifting green-emitting luciferases to the red end of the spectrum through multiple mutations in one single variant is common, with some achieving up to 90-100-nm shifts (reaching the emission peak of RE Ph ) 117 , inclusion of other mutations that, at the same time, stabilize and/or improve the activity is uncommon. Interestingly, in this case, the mutations that enhance the stability are not the same as those that shift colour; by contrast, the literature has numerous examples of mutations simultaneously affecting both 54,115,[118][119][120][121] . Nevertheless, to the best of our knowledge, no analysis has focused on using the plethora of mutations available for many luciferases yet (Figs 7,8). ...
In beetles, luciferase enzymes catalyse the conversion of chemical energy into light through bioluminescence. The principles of this process have become a fundamental biotechnological tool that revolutionized biological research. Different beetle species can emit different colours of light, despite using the same substrate and highly homologous luciferases. The chemical reasons for these different colours are hotly debated yet remain unresolved. This Review summarizes the structural, biochemical and spectrochemical data on beetle bioluminescence reported over the past three decades. We identify the factors that govern what colour is emitted by wild-type and mutant luciferases. This topic is controversial, but, in general, we note that green emission requires cationic residues in a specific position near the benzothiazole fragment of the emitting molecule, oxyluciferin. The commonly emitted green–yellow light can be readily changed to red by introducing a variety of individual and multiple mutations. However, complete switching of the emitted light from red to green has not been accomplished and the synergistic effects of combined mutations remain unexplored. The minor colour shifts produced by most known mutations could be important in establishing a ‘mutational catalogue’ to fine-tune emission of beetle luciferases, thereby expanding the scope of their applications.
... It provides high sensitivity, allowing for the detection of even subtle changes in microbial viability and metabolic activity. The assay provides real-time, noninvasive and continuous monitoring of antimicrobial effects [90,91], enabling researchers to observe dynamic changes in microbial populations during treatment. Furthermore, its quantitative nature allows for precise measurements of antimicrobial efficacy. ...
Infectious diseases pose a formidable global challenge, compounded by the emergence of antimicrobial resistance. Consequently, researchers are actively exploring novel antimicrobial compounds as potential solutions. This endeavor underscores the pivotal role of methods employed for screening and evaluating antimicrobial activity—a critical step in discovery and characterization of antimicrobial agents. While traditional techniques such as well-diffusion, disk-diffusion, and broth-dilution are commonly utilized in antimicrobial assays, they may encounter limitations concerning reproducibility and speed. Additionally, a diverse array of antimicrobial assays including cross-streaking, poisoned-food, co-culture, time-kill kinetics, resazurin assay, bioautography, etc., are routinely employed in antimicrobial evaluations. Advanced techniques such as flow-cytometry, impedance analysis, and bioluminescent technique may offer rapid and sensitive results, providing deeper insights into the impact of antimicrobials on cellular integrity. However, their higher cost and limited accessibility in certain laboratory settings may present challenges. This article provides a comprehensive overview of assays designed to characterize antimicrobial activity, elucidating their underlying principles, protocols, advantages, and limitations. The primary objective is to enhance understanding of the methodologies designed for evaluating antimicrobial agents in our relentless battle against infectious diseases. By selecting the appropriate antimicrobial testing method, researchers can discern suitable conditions and streamline the identification of effective antimicrobial agents.
... It provides high sensitivity, allowing for the detection of even subtle changes in microbial viability and metabolic activity. The assay provides real-time, non-invasive -26 -and continuous monitoring of antimicrobial effects [88,89], enabling researchers to observe dynamic changes in microbial populations during treatment. Furthermore, its quantitative nature allows for precise measurements of antimicrobial efficacy. ...
Infectious diseases pose a formidable global challenge, with the escalating threat of antimicrobial resistance complicating their treatment. In response, researchers are actively exploring natural and synthetic antimicrobial compounds as potential solutions. This pursuit of novel antimicrobial agents highlights the critical role of the methods used for screening and evaluating antimicrobial activity - a vital step in the discovery and characterization of effective antimicrobial compounds. While conventional techniques like well diffusion, disk diffusion and broth dilution methods are widely used in antimicrobial assays, they may face limitations in reproducibility and speed. Additionally, a variety of antimicrobial testing methods including cross-streaking, poisoned food, co-culture, time-kill kinetics, resazurin assay, bioautography, and more, are routinely employed in antimicrobial evaluation, expanding the range of assessment tools. Advanced techniques such as flow cytometry, impedance analysis and bioluminescent techniques may provide rapid and sensitive results, offering a deeper insight into the impact of antimicrobials on cellular integrity and viability, but their higher cost and limited accessibility in some laboratory settings can pose challenges. This article presents a comprehensive overview of the in-vitro and in-situ assays designed to characterize antimicrobial activity in natural and synthetic compounds, discussing the underlying principle, protocol, advantages, and limitations of each method. The primary objective is to enhance the understanding of available methods for evaluating antimicrobial agents in the ongoing battle against infectious diseases. By selecting the appropriate antimicrobial assay method, researchers can determine the most suitable conditions for assessment, and streamline the identification of effective antimicrobial agents.
... Currently, great progress has been made in the development of green fluorescent proteins, but the development of other colors of fluorescent proteins is in the early stages [8][9][10][11][12][13][14]. In addition, their short fluorescence lifetime and high requirements of the operation process are factors that limit their development [6]. ...
Carbon dots (CDs) are a strong alternative to conventional fluorescent probes for cell imaging due to their brightness, photostability, tunable fluorescence emission, low toxicity, inexpensive preparation, and chemical diversity. Improving the targeting efficiency by modulation of the surface functional groups and understanding the mechanisms of targeted imaging are the most challenging issues in cell imaging by CDs. Firstly, we briefly discuss important features of fluorescent CDs for live-cell imaging application in this review. Then, the newest modulated CDs for targeted live-cell imaging of whole-cell, cell organelles, pH, ions, small molecules, and proteins are elaborately discussed, and their challenges in these fields are explained.
Graphical abstract
... 282 As a thermoanalytical method, the technology can predict stability of biomolecules in various environmental conditions such as pH, ionic strength, and osmolites. 2,73,128,[283][284][285] Some types of this instrument can reveal volumetric features of the sample in different pressures. 11 In addition, the interaction of biomolecules and nanoparticles could be tracked via the calorimeters. ...
... Protein modification might result in the red or blue shift in the emitted color [29]. The in vitro bioluminescence spectra of wild type and circular LUC at pH 5.5 and pH 7.8, respectively, were obtained (25°C). ...
SpyTag can spontaneously form a covalent isopeptide bond with its protein partner SpyCatcher. Firefly luciferase from Photinus pyralis was cyclized in vivo by fusing SpyCatcher at the N terminus and SpyTag at the C terminus. Circular LUC was more thermostable and alkali-tolerant than the wild type, without compromising the specific activity. Structural analysis indicated that the cyclized LUC increased the thermodynamic stability of the structure and remained more properly folded at high temperatures when compared with the wild type. We also prepared an N-terminally and C-terminally shortened form of the SpyCatcher protein and cyclization using this truncated form led to even more thermostability than the original form. Our findings suggest that cyclization with SpyTag and SpyCatcher is a promising and effective strategy to enhance thermostability of enzymes.
ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.
Molecular dynamics (MD) at two temperatures of 300 and 340 K identified two histidine residues, His461 and His489, in the most flexible regions of firefly luciferase, a light emitting enzyme. We therefore designed four protein mutants H461D, H489K, H489D and H489M to investigate their enzyme kinetic and thermodynamic stability changes. Substitution of His461 by aspartate (H461D) decreased ATP binding affinity, reduced the melting temperature of protein by around 25 °C and shifted its optimum temperature of activity to 10 °C. In line with the common feature of psychrophilic enzymes, the MD data showed that the overall flexibility of H461D was relatively high at low temperature, probably due to a decrease in the number of salt bridges around the mutation site. On the other hand, substitution of His489 by aspartate (H489D) introduced a new salt bridge between the C-terminal and N-terminal domains and increased protein rigidity but only slightly improved its thermal stability. Similar changes were observed for H489K and, to a lesser degree, H489M mutations. Based on our results we conclude that the MD simulation-based rational substitution of histidines by salt-bridge forming residues can modulate conformational dynamics in luciferase and shift its optimal temperature activity.
Replacement of some hydrophobic solvent-exposed residues in Lampyris turkestanicus luciferase with arginine increases thermostability of this enzyme. Herein, thermodynamic and kinetic of unfolding reactions of wild type (WT), E354R/356R, E354R/356R-I232R and E354R/356R-Q35R/L182R/I232R variants has been investigated. Fluorescence and Far-UV circular dichroism signals using urea as chemical denaturant indicated that the value of ∆G(H2O) for all variants is greater than that of WT enzyme. Analysis of m-values, as a measure of difference in the solvent accessible surface area between the native and denatured states of protein, revealed that higher stability of mutants is related to their higher degree of compactness in the folded state. Results of unfolding kinetic experiments showed that all variants have three-exponential behavior in which, they unfolded with three rate constants and corresponding amplitudes. Increasing the rate constants of fast unfolding phase in mutants relative to WT protein may be attributed to more compactness and more kinetic sensitivity of their folded state to urea. However, more population of WT protein was unfolded from fast unfolding phase. Results of this investigation highlight kinetic stability of luciferase via a slow rate of unfolding. This article is protected by copyright. All rights reserved.
New and updated, this second edition of Protein Structure presents electrophoretic, chromatographic, and spectrophotometric techniques as well as including new chapters on the relatively sophisticated methods of mass spectrometry and ultracentrifugation. The emphasis is not on the technical aspects of the instruments, but more on the practical aspects of sample preparation and the possibilities of experimental strategy and design. The companion volume to this book, Protein Function: A Practical Approach, details comparable simple techniques for characterizing protein function. Topics in this second volume include: protein molecular weight determination by SDS-PAGE; protein analysis by mass spectrometry; immunological detection of proteins of known sequence; identification of common post-translational modifications; peptide mapping; counting integral numbers of residues by chemical modification; disulfide bonds between cysteine residues; analysis of protein conformation by gel electrophoresis; hydrodynamic properties of proteins; determining the molar absorbance coefficient of a protein; optical spectroscopy to characterize protein conformation and conformational changes; measuring the conformational stability of a protein; imunochemical analysis of protein conformation; stabilization of protein structure by solvents. Protein Structure: A Practical Approach will prove invaluable to all researchers, from PhD students to experienced group leaders.
As part of an effort to understand how proteins are imported into the peroxisome, we have sought to identify the peroxisomal targeting signals in four unrelated peroxisomal proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl-CoA oxidase. Using gene fusion experiments, we have identified a region of each protein that can direct heterologous proteins to peroxisomes. In each case, the peroxisomal targeting signal is contained at or near the carboxy terminus of the protein. For catalase, the peroxisomal targeting signal is located within the COOH-terminal 27 amino acids of the protein. For hydratase:dehydrogenase, D-amino acid oxidase, and acyl-CoA oxidase, the targeting signals are located within the carboxy-terminal 15, 14, and 15 amino acids, respectively. A tripeptide of the sequence Ser-Lys/His-Leu is present in each of these targeting signals as well as in the peroxisomal targeting signal identified in firefly luciferase (Gould, S.J., G.-A. Keller, and S. Subramani. 1987. J. Cell Biol. 105:2923-2931). When the peroxisomal targeting signal of the hydratase:dehydrogenase is mutated so that the Ser-Lys-Leu tripeptide is converted to Ser-Asn-Leu, it can no longer direct proteins to peroxisomes. We suggest that this tripeptide is an essential element of at least one class of peroxisomal targeting signals.
Scanning microcalorimetry and circular dichroism were used to study conformational state and heat denaturation of Ca2+-free synthetic calmodulin (SynCaM) and three charge reversal mutants. We produced evidence for the major role of the electrostatic potential in the stability and flexibility of SynCaM. The substitution of 118DEE120 by 118KKK120 (SynCaM12A) does not influence the flexibility of the protein; the replacement of 82EEE84 by 82KKK84 (SynCaM8) decreases its level, while the combination of these two mutations in SynCaM18A significantly increases the flexibility. The heat denaturation of apoSynCaM and its mutants is well approximated by two two-state transitions with the lower-temperature transition corresponding to C-terminal lobe melting and the higher-temperature one to N-terminal lobe melting. The difference in transition temperatures for the two lobes decreases in SynCaM8 and increases in SynCaM18A, suggesting a modification in the influence of one lobe to the other. The electrostatic mutations change the parameters of thermal denaturation of SynCaM lobes in a similar way as pH conditions affect thermal transition parameters of multidomain proteins, leading to a linear temperature dependence of transition enthalpy. One domain of the N-terminal lobe in apoSynCaM18A is unfolded in the native state. Near-UV CD spectra point out the invariability of the local structure of aromatic residues upon mutations, although the secondary structure undergoes striking transformations. Cacodylate ions strongly and specifically alter the helical content of SynCaM. Our data unambiguously demonstrate that the two lobes are not independent, and interactions between the lobes are mediated by the electrostatic potential of the molecule.
Abstract— Twenty-five Brazilian species (nine genera: Phorinus, Photinoides, Macrolampis, Aspisoma, Cratomorphus, Amydetes, Photuris, Bicellonychia, Pyrogaster) of adult fireflies were found to emit light in vivo in the green-yellow range (Λmax=548–573 nm) of the spectrum, more frequently near the green region, in contrast with North-American species, which predominantly emit yellow light. Distinct ecological contexts where these species evolved, such as the habitat (open field vs forests) and the duration of twilight, are discussed as possible factors responsible for these differences. Except for Photuris and Bicellonychia spp., the in vivo and in vitro bioluminescence spectra for various species of a given genus agree within ±5 nm. Lowering the pH caused the typical red shift in the in vitro bioluminescence spectrum from lampyrid luciferases (six species), which has been interpreted as due to the presence of a basic residue in the enzyme active site catalyzing fast enolization of the initially formed excited keto-oxyluciferin (red emitter) to the excited enol form (yellow-green emitter). The in vitro bioluminescence colors obtained from larval or adult elaterid (five species) and phengodid (three species) luciferases studied here, spanning the green-red region, do not respond to pH changes. This could indicate either the absence of the neighboring basic center (in red-emitting luciferases) or the presence of a non-pH affected proximal basic residue in the active site of the luciferase (in yellow-green-emitting luciferases).
Abstract— Bioluminescence, as a phenotype, has many evolutionary origins, and thus is an example of natural reinvention many times over. Although peculiar, it arises from the same biochemical principles and evolutionary mechanisms as other biochemical reactions. Of these many different bioluminescent systems, that of the luminous beetles is one of the best understood, having been extensively studied for over 50 years. The luminescence ensues from oxidation of a molecule unique to luminous beetles, beetle luciferin, through a catalytic mechanism evolved from ancestral coenzyme A synthetases. Thus, the character of this bioluminescent reaction is in part a consequence of that evolutionary history. Beetle bioluminescence is furthermore unusual in having a range of luminescent colors found among different beetle species and sometimes even within individual beetles. Structural features of the luciferases are responsible for these color differences, although the underlying mechanism is not yet clear.
IntroductionReactions Catalyzed by Firefly LuciferaseSubstrate Specificity and AnalogsPhysical Properties of Luciferase and Luciferase-Substrate ComplexNature of the Active SiteKinetics of Light Production and the Nature of the E-P ComplexLuciferase as an Activating Enzyme: Comparison with amino-acyl-tRNA SynthetasesSummary
All beetle luciferases have evolved from a common ancestor: they all use ATP, O2, and a common luciferin as substrates. The most studied of these luciferases is that derived from the firefly Photinus pyralis, a beetle in the superfamily of Cantharoidea. The sensitivity with which the activity of this enzyme can be assayed has made it useful in the measurement of minute concentrations of ATP. With the cloning of the cDNA coding this luciferase, it has also found wide application in molecular biology as a reporter gene. We have recently cloned other cDNAs that code for luciferases from the bioluminescent click beetle, Pyrophorus plagiophthalamus, in the superfamily Elateroidea. These newly acquired luciferases are of at least four different types, distinguishable by their ability to emit different colours of bioluminescence ranging from green to orange. Unique properties of these luciferases, especially their emission of multiple colours, may make them additionally useful in applications.